title: Ultrasensitive amplification refractory mutation system real-time PCR (ARMS RT-PCR) assay for detection of minority hepatitis B virus-resistant strains in the era of personalized medicine.
abstract: Resistance to antiviral treatment for chronic hepatitis B virus (HBV) has been associated with mutations in the HBV polymerase region. This study aimed at developing an ultrasensitive method for quantifying viral populations with all major HBV resistance-associated mutations, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) with a molecular beacon using a LightCycler. The discriminatory ability of this method, the ARMS RT-PCR with molecular beacon assay, was 0.01 to 0.25% for the different HBV resistance-associated mutations, as determined by laboratory-synthesized wild-type (WT) and mutant (Mut) target sequences. The assay showed 100% sensitivity for the detection of mutant variants A181V, T184A, and N236T in samples from 41 chronically HBV-infected patients under antiviral therapy who had developed resistance-associated mutations detected by direct PCR Sanger sequencing. The ratio of mutant to wild-type viral populations (the Mut/WT ratio) was >1% in 38 (63.3%) of 60 samples from chronically HBV-infected nucleos(t)ide analogue-naive patients; combinations of mutations were also detected in half of these samples. The ARMS RT-PCR with molecular beacon assay achieved high sensitivity and discriminatory ability compared to the gold standard of direct PCR Sanger sequencing in identifying resistant viral populations in chronically HBV-infected patients receiving antiviral therapy. Apart from the dominant clones, other quasispecies were also quantified. In samples from chronically HBV-infected nucleos(t)ide analogue-naive patients, the assay proved to be a useful tool in detecting minor variant populations before the initiation of the treatment. These observations need further evaluation with prospective studies before they can be implemented in daily practice.
authors.affiliation: Department of Hygiene, Epidemiology, and Medical Statistics, University of Athens Medical School, Athens, Greece.
authors.forename: Fotinie
authors.initials: F
authors.lastname: Ntziora
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Paraskevis
authors.forename: Catherine
authors.initials: C
authors.lastname: Haida
authors.forename: Emanuel
authors.initials: E
authors.lastname: Manesis
authors.forename: George
authors.initials: G
authors.lastname: Papatheodoridis
authors.forename: Spilios
authors.initials: S
authors.lastname: Manolakopoulos
authors.forename: Ioannis
authors.initials: I
authors.lastname: Elefsiniotis
authors.forename: Timokratis
authors.initials: T
authors.lastname: Karamitros
authors.forename: Alexis
authors.initials: A
authors.lastname: Vassilakis
authors.forename: Angelos
authors.initials: A
authors.lastname: Hatzakis
mesh_terms: D004279:DNA, Viral / Q000235:genetics*; D024882:Drug Resistance, Viral; D005838:Genotype; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000821:virology*; D006801:Humans; D020125:Mutation, Missense; D057285:Precision Medicine / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004279:DNA, Viral